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Effect of canal curvature location on the cyclic fatigue resistance of reciprocating files Sobotkiewicz, Tyler 2020

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EFFECT OF CANAL CURVATURE LOCATION ON THE CYCLIC FATIGUE RESISTANCE OF RECIPROCATING FILES by  Tyler Sobotkiewicz  DMD, University of Manitoba, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Craniofacial Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  February 2020  © Tyler Sobotkiewicz, 2020  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  Effect of canal curvature location on the cyclic fatigue resistance of reciprocating files.  submitted by Tyler Sobotkiewicz in partial fulfillment of the requirements for the degree of Master of Science in Craniofacial Science  Examining Committee: Dr. Ya Shen Supervisor  Dr. Markus Haapasalo Supervisory Committee Member  Dr. Ahmed Hieawy Supervisory Committee Member Dr. Vincent Lee Additional Examiner  iii  Abstract Objective: The aim of this study was to determine whether the cyclic fatigue resistance (CFR) of WaveOne (WO), WaveOne Gold (WOG), Reciproc (RE), and Reciproc Blue (REB) are affected by the location of the canal’s curvature. The phase transformation behaviors of the reciprocating file systems were also investigated. Methods: The reciprocating files were subjected to CFR testing in five artificial canals with a 60o angle of curvature and a 3 mm radius of curvature. The location of the curvature was unique for each canal. Each file was inserted 16 mm into the canal and operated until fracture occurred. The time to fracture was recorded and the length of the fractured fragment was measured. The surface of the fractured fragment was evaluated with scanning electron microscopy. Differential scanning calorimetry was used to characterize the file’s thermal behavior. Results: Reciprocating files have significantly decreased CFR in canals with middle- and coronally-located curvatures compared to those with apically-located curvatures (p < 0.05). At all tested canal curvature locations, REB had significantly superior CFR compared to WO and RE (p < 0.05). There were no significant differences in CFR between WOG and REB in canals with middle- or coronally-located curvatures (p > 0.05).  There were no significant differences in the fractured fragment length between the file systems (p > 0.05) except between WOG and RE in the canal with an orifice to curvature distance of 11 mm (p < 0.05). Surface characterization of the surface of the fractured fragments showed typical patterns of cyclic fatigue failure.  Conclusion: The location of the canal curvature had a significant effect on the CFR of the reciprocating files. WOG and REB are more resistant to cyclic fatigue than WO and RE. When heated, WO, WOG, and RE underwent a one-stage phase transformation, while REB underwent a two-stage phase transformation. iv  Lay Summary The goal of root canal treatment is to eliminate microbes in root canals and seal the tooth in order to resolve the signs and symptoms of endodontic disease. The combination of mechanical debridement with nickel-titanium rotary files and chemical disinfection with irrigants (such as sodium hypochlorite) are used to reduce the intra-canal bacterial load. Rotary files, which can be used in continuous rotation or reciprocation movements, are at risk of fracture when used in canals that are curved. Endodontic disease may persist after root canal treatment if fractured files block and prevent further disinfection in the root canal. The objective of this study was to determine whether the location of the root canal’s curvature influences the fracture resistance of rotary files that are used with reciprocating movements.  v  Preface This study was approved by the University of British Columbia Clinical Research Ethics Board (H12-02430). This thesis is the principle work of the candidate, Tyler Sobotkiewicz, as per the requirements of a Master of Science in Craniofacial Science with a Diploma in Endodontics.  Tyler Sobotkiewicz was responsible for performing the fatigue tests, preparing the file fragments for SEM, performing the DSC, interpretation and statistical analysis of the results, and writing of the thesis. The supervisor of this project, Dr. Ya Shen, contributed to the design of the study, analysis of the results, and editing of the thesis. Support and consultation were provided by Dr. Ahmed Hieawy and Dr. Markus Haapasalo. Jinghao Hu contributed to this project by obtaining the SEM images and providing support for statistical analysis.  vi  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiii Dedication ................................................................................................................................... xiv Chapter 1: Introduction ................................................................................................................1 1.1 Goal of endodontics ........................................................................................................ 1 1.2 Nickel-titanium instruments............................................................................................ 2 1.2.1 Generations of nickel-titanium rotary files ................................................................. 4 1.2.2 Instrument fracture ...................................................................................................... 6 1.2.2.1 Stages of fatigue failure ...................................................................................... 8 1.2.2.2 Clinical implications of rotary file fracture ...................................................... 10 1.2.2.3 Cyclic fatigue testing apparatuses ..................................................................... 11 1.3 Differential scanning calorimetry (DSC) ...................................................................... 13 1.4 Rationale ....................................................................................................................... 13 1.5 Aims .............................................................................................................................. 14 1.6 Null hypothesis ............................................................................................................. 14 vii  Chapter 2: Materials and Methods ............................................................................................15 2.1 Sample size calculation ................................................................................................. 15 2.2 Cyclic fatigue test ......................................................................................................... 15 2.3 Differential scanning calorimetry ................................................................................. 21 2.4 Statistical analysis ......................................................................................................... 23 Chapter 3: Results........................................................................................................................24 3.1 Cyclic fatigue test ......................................................................................................... 24 3.1.1 Differences based on file system .............................................................................. 24 3.1.2 Differences based on canal curvature location ......................................................... 24 3.1.3 Fractured fragment length ......................................................................................... 27 3.1.4 Fractographic examination........................................................................................ 29 3.2 Differential scanning calorimetry ................................................................................. 34 Chapter 4: Discussion ..................................................................................................................40 Chapter 5: Conclusion .................................................................................................................46 Bibliography .................................................................................................................................47 viii  List of Tables  Table 1: Number of cycles to failure (NCF). Statistically significant differences (p < 0.05) exist between groups in a row that have different superscript numbers, and when values in a column have different subscript letters. Canal groups: 1, 2, 3, 4, and 5 represent 5 mm, 6 mm, 8 mm, 10 mm, and 11 mm distance between the canal orifice and the curvature location (DOC), respectively. .................................................................................................................................. 25 Table 2: The length of the fractured fragments in millimeters. Statistically significant differences (p < 0.05) exist between groups in the same row with different subscript letters. ....................... 27 Table 3: Mean phase transformation temperatures + standard deviation acquired by cooling the apical and coronal fragments of the reciprocating file systems. Ms = martensite transformation start temperature. Mf = Martensite transformation finish temperature. There were no significant differences in Ms or Mf between the apical and coronal fragments of each file system (p > 0.05)........................................................................................................................................................ 35 Table 4: Mean phase transformation temperatures + standard deviation acquired by heating the apical and coronal fragments of the reciprocating file systems. Rs = R-phase transformation start temperature. As = Austenite transformation start temperature. Af = Austenite transformation finish temperature. There were no significant differences in Rs, As or Af between the apical and coronal fragments of each file system (p > 0.05).......................................................................... 36 Table 5: Mean phase transformation temperatures + standard deviation of the reciprocating file systems when the coronal and apical fragments were combined into one group. ........................ 36  ix  List of Figures  Figure 1: Photograph of NiTi reciprocating files (from left to right): WaveOne, WaveOne Gold, Reciproc, Reciproc Blue. ................................................................................................................ 6 Figure 2: SEM image of the surface of a fractured rotary file due to cyclic fatigue failure. A: The crack initiation point is circled. B: Higher magnification image of A. Note fatigue striation lines (arrow). Courtesy of Dr. Ya Shen. ......................................................................................... 9 Figure 3: SEM image of the surface of a fractured rotary file due to shear (torsional) failure. Note the relatively smooth surface of the fractured fragment surface and lack of fatigue striation lines. Courtesy of Dr. Ya Shen. ...................................................................................................... 9 Figure 4: Sample calculation for Number of Cycles to Failure (NCF) for WaveOne and WaveOne Gold. TTF is the Time to Fracture in seconds. ............................................................ 18 Figure 5: Sample calculation for Number of Cycles to Failure (NCF) for Reciproc and Reciproc Blue. TTF is the Time to Fracture in seconds. .............................................................................. 18 Figure 6: Photograph of the experimental set-up......................................................................... 19 Figure 7: Photograph of a mounted WaveOne Gold rotary file inside an artificial canal. .......... 19 Figure 8: Schematic diagrams of the artificial canals (A: Group 1; B: Group 2; C: Group 3; D: Group 4; E: Group 5), and a photograph of the zirconium oxide block containing the five artificial canals (F). ....................................................................................................................... 20 Figure 9: Photograph of the DSC2500 machine. ......................................................................... 22 Figure 10: Photograph of DSC2500 tray containing samples that are sealed in aluminum cells. 22 x  Figure 11: Comparison of the mean NCF between different reciprocating NiTi rotary files when used in artificial canals with different curvature locations. Statistically significant differences (p < 0.05) exist between groups with different letters....................................................................... 26 Figure 12: Effect of canal curvature location on the mean NCF of reciprocating NiTi rotary files. Statistically significant differences (p < 0.05) exist between groups with different letters. ......... 26 Figure 13: Comparison of the mean fractured fragment length between different reciprocating NiTi file systems in artificial canals with different canal curvature locations. Statistically significant differences (p < 0.05) exist between groups with different letters.............................. 28 Figure 14: Effect of the canal curvature location on the mean fractured fragment length of reciprocating NiTi reciprocating file systems ............................................................................... 28 Figure 15: SEM fractograph (x150) of a WO file fragment. Crack initiation points are shown by white arrows. ................................................................................................................................. 30 Figure 16: Increased magnification (x600) of Figure 15. Crack initiation points are shown by white arrows and fatigue striations are shown by black arrows. .................................................. 30 Figure 17: SEM fractograph (x250) of a WOG file fragment. Crack initiation points are shown by white arrows. ............................................................................................................................ 31 Figure 18: Increased magnification (x650) of Figure 17. Crack initiation points are shown by white arrows. ................................................................................................................................. 31 Figure 19: SEM fractograph (x130) of a RE file fragment. Crack initiation points are shown by white arrows. ................................................................................................................................. 32 Figure 20: Increased magnification (x550) of Figure 19. Crack initiation points are shown by white arrows and fatigue striations are shown by black arrows. .................................................. 32 xi  Figure 21: SEM fractograph (x130) of a REB file fragment. Crack initiation points shown by white arrows. ................................................................................................................................. 33 Figure 22: Increased magnification (x500) of Figure 21. Crack initiation points shown by white arrows and fatigue striations are shown by black arrows. ............................................................ 33 Figure 23: DSC thermogram comparing the apical and coronal fragments of WO. ................... 37 Figure 24: DSC thermogram of a WO coronal fragment. ........................................................... 37 Figure 25: DSC thermogram of a WOG coronal fragment. ........................................................ 38 Figure 26: DSC thermogram of a RE coronal fragment. ............................................................. 38 Figure 27: DSC thermogram of a REB coronal fragment. .......................................................... 39 Figure 28: DSC thermogram comparing the coronal fragments of WO, WOG, RE, and REB. . 39  . xii  List of Abbreviations ADA: American Dental Association ANSI: American National Standards Institute  As = Austenite transformation start temperature Af = Austenite transformation finish temperature CFR: Cyclic fatigue resistance DSC: Differential scanning calorimetry EDTA: Ethylenediaminetetraacetic acid  ISO: International Organization for Standardization Ms = Martensite transformation start temperature Mf = Martensite transformation finish temperature  NCF: Number of cycles to failure NiTi: Nickel titanium RE: Reciproc REB: Reciproc Blue rpm = Rotations per minute Rs = R-phase transformation start temperature SD: Standard deviation SEM: Scanning electron microscope TTF: Time to fracture WO: WaveOne WOG: WaveOne Gold  xiii  Acknowledgements Firstly, I would like to thank my supervisor, Dr. Ya Shen, for graciously sharing her knowledge, wisdom, and positive energy with me for the past two years as I worked on this research project and studied endodontics at the University of British Columbia. I am grateful to my committee members, Dr. Ahmed Hieawy and Dr. Markus Haapasalo for always being available and willing to go the extra mile when I needed assistance. Furthermore, I would like to thank Jinghao Hu for providing support in the lab as well as Christophe Mobuchon and Sherry Kiafar who provided direction regarding the use of DSC and TRIOS software.  I am very thankful for the mentorship that has been provided by the director of the program, Dr. Jeff Coil, as well as our clinical instructors. Lastly, I would like to thank my grad endo co-residents, and our clinic CDAs, Shauna, Lois, and Francisco, for their help, support, and companionship throughout the years.  xiv  Dedication I dedicate this work to my family who have encouraged and supported me throughout my personal and academic pursuits. I would also like to thank my friends who have made every effort to keep me motivated as I accomplish my professional goals and for providing healthy distraction, when needed. 1  Chapter 1: Introduction 1.1 Goal of endodontics Endodontic disease is characterized by a bacterial infiltration of the pulp resulting in pulpitis, pulpal necrosis, and/or apical periodontitis (Kakehashi et al. 1965, Sundqvist 1976). The goal of endodontic treatment is to eliminate intra-canal microorganisms and pathologic pulp tissue in order to resolve the signs and symptoms related to endodontic disease. Root canal treatment includes cleaning and shaping in order to disinfect and prepare the canals for obturation. Obturation/filling of the canal is performed in order to entomb bacteria that survived the cleaning and shaping phase while creating a seal in an attempt to prevent future bacterial infiltration and re-infection of the root canal system (Johnson et al. 2016). Finally, a permanent coronal restoration is essential after root canal treatment in order to seal both the dentin and root canal system from bacteria in the oral cavity.  Cleaning and shaping is also described as chemo-mechanical preparation. Mechanical preparation involves the use of hand-held and engine-driven rotary files that debride dentin from the canal walls. Due to the naturally irregular morphology of root canals, rotary instrumentation leaves up to 34.6% of root canal walls untouched (Siqueira Jr et al. 2018, Zuolo et al. 2018). Undisturbed and viable bacterial biofilm on untouched canal walls can be a source of persistent apical periodontitis (Siqueira Jr et al. 2012). Bystrom and Sundqvist found that mechanical debridement with saline irrigation of necrotic root canals was capable of reducing intracanal bacterial numbers by 102-103, but this protocol was inconsistent as they were able to recover bacteria in 47% of canals at the end of treatment (Bystrom & Sundqvist 1981). Mechanical preparation combined with sodium hypochlorite irrigation has been shown to significantly 2  reduce bacterial numbers when compared to mechanical preparation with saline (Bystrom & Sundqvist 1983). Sodium hypochlorite is commonly used as an irrigant in endodontics due to its tissue-dissolving and antimicrobial properties (Siqueira et al. 1997, Jeansonne & White 2006, Stojicic et al. 2010). Mechanical canal enlargement is important as it can improve irrigant penetration and renewal throughout the canal system, which can maximize the antimicrobial effect of sodium hypochlorite (Bronnec et al. 2010). Due to their synergistic benefits, the combination of adequate mechanical preparation and chemical disinfection has been imperative in root canal treatment.  1.2 Nickel-titanium instruments Walia et al. introduced nickel titanium (NiTi) hand files, which had greater flexibility and superior resistance to torsional fracture, when compared to traditional stainless steel files (Walia et al. 1988). The composition of endodontic NiTi instruments is 56% nickel and 44% titanium by weight, resulting in a 1:1 atomic ratio between the elements (Shen et al. 2013). The crystal structure of NiTi can exist in three different states (austenite, martensite, and R-phase), which influences the physical properties of the alloy. Notable features of NiTi include superelasticity and shape memory. Superelasticity is the ability of an alloy to revert to its original shape once the external stresses, which caused its deformation, are relieved (Otsuka & Ren 2005). The ability of a NiTi instrument to return to its original shape once heat is applied is defined as shape memory (Otsuka & Ren 2005). Temperature and stress influence whether the NiTi will exist in an austenitic, martensitic, or R-phase. 3  The austenite phase exists at higher temperatures and is stronger and stiffer than the martensite phase, which exists at lower temperatures and is softer and more flexible (Shen et al. 2013). When martensitic NiTi is heated past the austenite transformation start temperature (As), the NiTi’s crystal structure begins to change to austenite and will become fully austenite once heated past the austenite transformation finish temperature (Af). The heat-induced transformation of martensite to austenite is the basis of NiTi’s shape memory characteristic. Conversely, as austenite cools it will begin to change to martensite at the martensite transformation start temperature (Ms) and will complete the transition to martensite at the martensite transformation finish temperature (Mf). The phase transformation from austenite to martensite can also be stress-induced, as seen in superelastic NiTi alloys, which allows the NiTi to accommodate a greater amount of stress without an increase of strain (Thompson 2000, Otsuka & Ren 2005). The martensite to austenite and austenite to martensite phase transformations can occur in either one or two stages, where the two-stage transformation involves the formation of an intermediate R-phase.(Otsuka & Ren 2005) The R-phase is a temperature-induced or stress-induced intermediate phase that demonstrates shape memory and superelasticity (Zhou et al. 2013, Santos et al. 2016). The R-phase has a relatively low Young’s modulus resulting in the R-phase being more flexible than martensite (Zhou et al. 2013, Santos et al. 2016). Conventionally, NiTi instruments existed in the austenite phase at room and body temperature, which limits their use in severely curved canals due to the instrument’s stiffness and low fatigue resistance (Brantley et al. 2002). Heat treatment produces a more flexible and fatigue resistant NiTi instrument by releasing the internal strain of the alloy and increasing the NiTi’s phase 4  transformation temperatures, resulting in a more martensitic alloy at clinically relevant temperatures (Kuhn & Jordan 2002, Otsuka & Ren 2005).   1.2.1 Generations of nickel-titanium rotary files The first generation of NiTi rotary files included files with radial lands, such as LightSpeed and ProFile 0.04 file series. Radial lands are flat surfaces that exist between 2 flutes of the file and are useful in reducing canal transportation by keeping the file centered in the canal, but it is at the expense of cutting efficiency (Maitin et al. 2013). Second generation files are characterized by active cutting edges and usually lack radial lands. ProTaper Universal and Race files are an example of second generation files that have increased cutting efficiency compared to first generation files due to their triangular cross-section. The third generation of NiTi rotary files have improved metallurgy due to thermomechanical processing by the manufacturers. The objective of thermomechanical processing is to modify the phase transformation temperatures of the NiTi alloy so that the file is more flexible and less prone to fatigue failure during clinical use (Frick et al. 2005, Yahata et al. 2009,). M-wire NiTi files, such as ProFile Vortex and Vortex Blue, are made from NiTi wire blanks that were exposed to a series of heat-treatments during processing. Meanwhile, K3XF files are plastically deformed during manufacturing by a process that includes the twisting of the file as it is exposed to specific thermal conditions. Rotary files made from Controlled Memory wire (eg. HyFlex CM) are unique as they are extremely flexible and lack shape memory, allowing their relatively safe use in complex canal anatomy (Shen et al. 2013). 5  The fourth generation of files are used in a reciprocation movement in contrast to the continuous rotational movement used by the other generations. Yared was the first author to report the use and efficiency of reciprocating movements for rotary files, as he demonstrated that a single reciprocating file was sufficient to replace a series of multiple files used in continuous rotation (Yared 2007). Reciprocating files engage and debride dentin when rotating in the cutting direction and will disengage in the opposite direction. The benefits of reciprocation include decreased formation of intra-dentinal cracks and decreased risk of fatigue file fracture, when compared to continuous rotation (De Deus et al. 2010, Pedulla et al. 2013, Karatas et al. 2015). Reciprocating movements result in the rotary file experiencing fewer complete rotations and fewer tensile-compressive stress cycles compared to continuous rotation, which may account for the increased cyclic fatigue resistance (De Deus et al. 2010). WaveOne (WO) (Dentsply Sirona, York, Pennsylvania, USA) and Reciproc (RE) (VDW Munich, Germany) are among the earliest manufactured NiTi rotary files intended for reciprocating movements. Years later, heat treated reciprocating files, WaveOne Gold (WOG) (Dentsply Sirona) and Reciproc Blue (REB) (VDW Munich), were released and demonstrated increased flexibility compared to their predecessors (Prados-Privado et al. 2019). WOG and REB have been shown to be a safer alternative to WO and RE in curved canals due to their relatively superior cyclic fatigue resistance (Özyürek 2016, Keskin et al. 2017, Al-Obaida et al. 2019, Scott et al. 2019, Keles et al. 2019). Rotary instruments that comprise the fifth generation, such as ProTaper Next and One Shape, are manufactured with an off-set center of mass and/or rotation. An off-set design minimizes the contact surface area between the file and the dentin at a single point in time, decreasing the risk of taper lock and the amount of torque on the file (Haapasalo & Shen 2013). 6    Figure 1: Photograph of NiTi reciprocating files (from left to right): WaveOne, WaveOne Gold, Reciproc, Reciproc Blue.  1.2.2 Instrument fracture While rotary instrumentation results in less canal transportation, less dentin removal, and less treatment time than hand instrumentation, there has been concern regarding rotary file fracture (Glosson et al. 1995, Tharuni et al. 1996). Endodontic rotary instruments fracture due to torsional overloading or cyclic fatigue. If an instrument continues to rotate while its tip is bound in the canal, the shear strength of the NiTi may be exceeded and will result in fracture due to torsional overloading (also known as shear failure) (Cheung 2009). Files subject to torsional overloading will demonstrate signs of defects, such as unwinding, that occur near or within several millimeters of where the fracture will occur (Sattapan et al. 2000). As unwinding is visible to the naked eye, it is important for the clinician to be vigilant in regularly inspecting rotary files during treatment and discarding files with defects in order to prevent file fracture due to shear failure.  7  Fracture due to cyclic fatigue occurs when an instrument freely rotates in a curved canal. The half of the file at the inside of the curve undergoes compressive stress while the half on the outside of the curve experiences tensile stresses (Pruett et al. 1997). The rotary file will endure one complete tension-compression stress cycle with each rotation it completes in a curved canal. Unlike torsional overloading, files subject to cyclic fatigue will not demonstrate visible signs of defects and the fracture point will occur at the canal’s point of maximum curvature (Pruett et al. 1997, Sattapan et al. 2000). The risk of cyclic fatigue failure of a NiTi rotary file is affected by the anatomy of the root canal and the file’s cyclic fatigue resistance (CFR), which is dependent on its metallurgy and design. Geometric properties of canals that increase the risk of fatigue failure include small radii of curvature, large angles of curvature, and curvatures with long arc lengths (Pruett et al. 1997, Haïkel et al. 1999, Lopes et al. 2013). Additionally, canals with curvatures located closer to the canal orifice and canals with double curvatures (S-shaped canals) have been shown to increase the risk of fatigue failure of rotary files (Lopes et al. 2011, Al-Sudani et al. 2012). Rotary files with a larger diameter and/or taper will have decreased CFR when compared to rotary files with smaller diameters and/or tapers (Pruett et al. 1997, Haïkel et al. 1999). As evidence of cyclic fatigue cannot be gathered by visual inspection of the file during treatment, it is important for clinicians to choose a rotary file system with an appropriate CFR for the root canal anatomy at hand. Additional ways to reduce the risk of fatigue and shear failure include the creation of a glide path, avoiding excessive apical pressure during instrumentation, avoiding the over-use of rotary files, and the use of a torque-controlled motor and lubrication during mechanical preparation of the canal.  8  1.2.2.1 Stages of fatigue failure Pruett et al. were able to demonstrate three stages of fatigue failure by using scanning electron microscopy (SEM) on the instrument’s fractured surface (Pruet et al. 1997). The first stage, crack initiation and growth, is characterized by a smooth area at the periphery of the fractured file’s surface. Cracks typically originate near the cutting edge or at defects on the instrument’s surface, such as machining grooves (Cheung et al. 2007, Lopes et al. 2011). The second stage, crack propagation, is seen as striations that begin from the periphery and grow towards the center of the instrument. Fatigue striations, which represent progression of the crack, are created as the instrument continues to experience tensile-compressive stress cycles. The third stage, the ultimate ductile fracture, is characterized by the formation of a microvoid and dimpling in the center of the fracture surface.    9   Figure 2: SEM images of the surface of a fractured rotary file due to cyclic fatigue failure. A: The crack initiation points are indicated by arrows. B: Higher magnification image of A. Note fatigue striation lines (arrows). Image from Shen et al. (Shen et al. 2009), by permission from publisher.   Figure 3: SEM image of the surface of a fractured rotary file due to shear (torsional) failure. Note the relatively smooth surface of the fractured fragment surface and lack of fatigue striation lines. Image from Cheung et al. (Cheung et al. 2007), by permission from publisher.  A B 10  1.2.2.2 Clinical implications of rotary file fracture The overall prevalence of fractured instruments during root canal treatment has been found to range between 1.7-3.3%, with rotary files responsible for 70-85% of the fractured instruments (Spili et al. 2005, Iqbal et al. 2006, Tzanetakis et al. 2008). Using microscopic and fractographic analysis, authors have concluded that 66-93% of rotary file fractures were due to cyclic fatigue failure while 7-34% were due to shear failure (Cheung et al. 2005, Peng et al. 2005, Shen et al. 2009). Clinicians may choose to either bypass or remove fractured instruments to regain access to the complete extent of the root canal. Due to difficult and complex canal anatomy, such as thin or curved roots, the clinician may elect to leave the fractured instrument in the canal when there is a risk of perforation and/or excessive dentin removal. In cases where the fractured instrument cannot be removed or bypassed, it is unlikely that there will be further chemo-mechanical disinfection or obturation in the apical portion of the root canal. In necrotic teeth with bacteria remaining beyond the broken instrument, it is to be expected that these teeth would have persistent periapical disease and thus a poor prognosis for root canal treatment. Amazingly, studies have shown no significant differences in success of root canal treatment in teeth with retained fractured instruments and those without (Crump & Natkin 1970, Spili et al. 2005, Panitvisai et al. 2010). Although there appears to be no significant decrease in prognosis, fractured instruments should be avoided if possible, to assure complete chemo-mechanical disinfection and obturation of the canal, and to avoid psychological suffering in patients.   11  1.2.2.3 Cyclic fatigue testing apparatuses  Standardized tests have been developed to measure the torsional resistance and flexibility of stainless steel hand files (ANSI/ADA specification No. 28) and rotary instruments (ISO 3630-1). Currently, there is no standardized test for measuring the CFR of endodontic rotary files. Plotino et al. has reviewed apparatuses that have been used to study the CFR of rotary files, including curved metal tubes, grooved and sloped metal blocks, three-point bending systems using steel pins, and artificial/simulated canals (Plotino et al. 2009). Metal tubes, formed by bending 18 gauge stainless steel needles or stainless steel tubing have been used for CFR testing (Pruett et al. 1997, Chaves et al. 2002). These metal tubes have a constant inner diameter and are not able to restrict the shaft of the rotary instrument. During rotation, the rotary instrument may act unpredictably and follow a trajectory of greater radius of curvature and reduced angle of curvature, which reduces consistency between samples and overestimates the CFR of the tested rotary files (Chaves et al. 2002). Sloped metal blocks with a 2 mm groove for the tip of the instrument have been used but were found unreliable as the desired angle of curvature could not be accurately reproduced between different file systems (Plotino et al. 2009). An apparatus consisting of three stainless steel pins, which constrained the instrument into the desired curvature has been used (Cheung et al. 2007). If not constrained properly between the steel pins, rotary instruments of different systems may follow different trajectories due to differences in flexibility, taper, and/or cross-sectional design (Plotino et al. 2009).  Artificial canals, created by grooving a stainless-steel block and attaching a piece of tempered glass, can provide consistent trajectories for fatigue resistance tests as the groove can be 12  customized for the rotary file’s taper and diameter. Plotino et al. compared the cyclic fatigue resistance of ProFile and Mtwo in two stainless steel artificial canals that differed only in diameter (Plotino et al. 2010). This study concluded that rotary files operating in artificial canals customized 0.1 mm larger than the file resulted in a significantly more predictable trajectory than files rotating in canals that were customized 0.3 mm larger (Plotino et al. 2010). Currently, artificial canals appear to be the best apparatus available for accurate CFR testing. A downside of artificial canals is that the friction between the rotating file and the walls of the artificial canal may be able to create enough heat to cause phase transformations of the NiTi instrument (Haïkel et al. 1999).  Lubricating agents have been employed in artificial canals to reduce friction between the rotating file and the canal walls and to allow control over the environmental temperature. Media that have been used for cyclic fatigue testing include air (no media), water, synthetic oil, glycerin, sodium hypochlorite, and ethylenediaminetetraacetic acid (EDTA). Shen et al. found that files rotating in curved canals filled with aqueous media, such as water or EDTA, took significantly longer to experience cyclic fatigue failure when compared to unfilled canals (air) (Shen et al. 2012). The author concluded that the aqueous media may act as a “heat-sink”, in that it disperses the frictional heat created between the rotating file and the canal walls and reduces the risk of a martensite to austenite phase transformation (Shen et al. 2012). Other studies have shown that rotary files operating in canals filled with synthetic oil or air have a significantly superior fatigue life than when they are operated in canals with water or sodium hypochlorite, possibly due to the corrosive nature of these aqueous mediums (Cheung et al. 2007, Elnaghy & Elsaka 2016). The results of these studies suggest that it may be important for future fatigue resistance studies to 13  use media that replicates clinical root canal treatment in order to have the most clinically relevant results.  1.3 Differential scanning calorimetry (DSC) The phase transformations of NiTi are temperature-dependent and thus there will be a change in enthalpy as the NiTi transitions between phases. The austenite to martensite transformation involves an exothermic reaction while the martensite to austenite transformation involves an endothermic reaction. Differential scanning calorimetry makes use of a controlled environment where the experimental subject and a control are heated or cooled at the same rate. As the experimental subject undergoes phase transformation, which involves an exothermic or endothermic reaction, DSC can measure minute differences in enthalpy between the experimental subject and the control. The data gained from DSC can be used to determine the temperatures at which the NiTi will begin and finish phase transformations (Shen & Cheung 2013, Hieawy et al. 2015, Shen et al. 2015).   1.4 Rationale Reciproc, Reciproc Blue, WaveOne, and WaveOne Gold have different metallurgy, cross-sectional designs, taper, and manufacturer’s recommended use, which may affect their fatigue resistance in different canal anatomies. While fatigue resistance studies of these reciprocating file systems are available, there is a gap in the literature concerning the effect of the canal’s curvature location on the CFR of these rotary files.  14  Since the physical properties of NiTi are affected by the environmental temperature, it is important to characterize the phase transformation temperatures of the reciprocating file systems. Knowing the NiTi rotary file’s phase and physical properties at body temperature may aide the clinician in selecting an adequate rotary file system for the particular endodontic case at hand.   1.5 Aims • To examine the effect that the location of the canal’s curvature has on the CFR of reciprocating NiTi files.  • To examine and compare the CFR profiles of RE, REB, WO, and WOG. • To analyze the thermal behavior and phase transformation temperatures of the apical and coronal portions of RE, REB, WO, and WOG using DSC.  1.6 Null hypothesis The CFR of reciprocating files is not affected by the location of the canal’s curvature. There is no difference in the CFR of RE, REB, WO, and WOG file systems. There are no significant differences in the phase transformation temperatures between the apical and coronal portions of the rotary files. 15  Chapter 2: Materials and Methods This study was approved by the University of British Columbia Clinical Research Ethics Board (H12-02430).  2.1 Sample size calculation Sample size was determined using G*Power version 3.1.9.4 software (Brunsbuttel, Germany). A paper by Ozyurek that studied the CFR of reciprocating instruments was used to calculate the effect size while the significance level was set at 0.05 and power was set at 0.80 (Ozyurek 2016). The software calculated that 8 files per group were necessary, and the decision was made to include 10 files per group. As there are 20 groups, 200 total files (50 of each file system) were used for the experiment.    2.2 Cyclic fatigue test Five ceramic artificial canals were milled in an InCoris ZI zirconium oxide disc (Dentsply Sirona) using the inLab MC X5 Digital computer-aided design and computer-aided manufacturing (CAD/CAM) System (Dentsply Sirona). Each artificial canal was 16 mm in length and was represented by a tapered groove on one side of the zirconium oxide block. The most coronal extent of the canal, the canal orifice, was 1.31 mm in diameter and constantly tapered by 0.06 mm per every millimeter of canal length, resulting in a 0.35 mm diameter for the apical extent of the canal. Each canal had a single curvature with a radius of curvature of 3 mm and an angle of curvature of 60o, as defined by Weine’s method (Balani et al. 2015). The five artificial canals differed only in the location of their curvatures along the length of the canal. The distance between the canal orifice to the location of the curvature will be abbreviated as DOC 16  (Distance from the Orifice to the Curvature). The canals were separated into 5 groups, based on the location of where the curvature began; Group 1: DOC of 5 mm; Group 2: DOC of 6 mm; Group 3: DOC of 8 mm; Group 4: DOC of 10 mm; Group 5: DOC of 11 mm. Schematic diagrams and a photograph of the zirconium oxide block containing the artificial canals can be seen in Figure 8. A piece of fiber glass was secured to the grooved side of the zirconium oxide blocks to retain the rotary files within the artificial canal. The artificial canal was placed inside a glass container that was filled with deionized water. Within the glass container, plastic plumber screws were used to stabilize the fiber glass to the zirconium oxide block. The glass container was placed on a hotplate (Radnor, Pennsylvania, USA), which kept the deionized water at a constant temperature of 37 + 2oC. A red-spirit thermometer was left inside the glass container and was used to monitor the deionized water’s temperature throughout the experiment. A photograph of the experimental set-up is shown in Figure 6. The following NiTi rotary files were subject to cyclic fatigue testing: A) WO Primary (25/.08)  B) WOG Primary (25/.07)  C) RE-25 (25/.08)  D) REB-25 (25/.08)  A total of 50 files of each file system were used and all files were 25 mm in length. Each file system was evenly distributed to the five groups of canals. This resulted in a total of 20 groups, which included 10 files of each file system being tested in each of the artificial canals. A stainless-steel framework that was attached to the hotplate was used to secure the motor 17  handpiece in position. RE and REB files were used with a 6:1 reduction handpiece connected to a VDW Silver motor (VDW Munich). WO and WOG files were used with an 8:1 reduction handpiece connected to a ProMark Endo motor (Dentsply Sirona). As per the manufacturer’s instructions, RE and REB were operated with “RECIPROC ALL” settings while WO and WOG were operated with “WAVEONE ALL” settings. The rotary file was placed to the full length of the artificial canal (Figure 7) and was operated until fracture occurred. Each specimen was recorded with a video camera that was capable of high definition magnification (Panasonic, Mississauga, Ontario, Canada), which was reviewed to determine the time to fracture (TTF) in seconds. The number of cycles to fracture (NCF) was calculated by dividing the TTF by 60 seconds/minute and multiplying by the number of rotations per minute (rpm) that each file operates at. RE and REB operate at 300 rpm (via the “RECIPROC ALL” setting) while WO and WOG operate at 350 rpm (via the “WAVEONE ALL” setting) (Ozyurek 2016, Yılmaz & Ozyurek 2017). Figure 4 and Figure 5 show sample formulae for calculating the NCF for the WaveOne systems and Reciproc systems, respectively.  Each fractured fragment was collected and its length was measured under magnification by means of a surgical operating microscope (Global, St. Louis, Missouri, USA). Two fragments of each file system per group were randomly chosen for fractographic examination. The fragments were submerged in absolute alcohol and ultrasonically activated (Endo Ultra, MicroMega, Besancon, France) for 60 seconds. The fragments were mounted with the fractured surface facing upwards and were submitted for SEM (Helios Nano Lab 650; FEI, Eindhoven, Netherlands).  18    Figure 4: Sample calculation for Number of Cycles to Failure (NCF) for WaveOne and WaveOne Gold. TTF is the Time to Fracture in seconds.     Figure 5: Sample calculation for Number of Cycles to Failure (NCF) for Reciproc and Reciproc Blue. TTF is the Time to Fracture in seconds.19   Figure 6: Photograph of the experimental set-up.  Figure 7: Photograph of a mounted WaveOne Gold rotary file inside an artificial canal.20    Figure 8: Schematic diagrams of the artificial canals (A: Group 1; B: Group 2; C: Group 3; D: Group 4; E: Group 5), and a photograph of the zirconium oxide block containing the five artificial canals (F).   A B C D E F 21  2.3 Differential scanning calorimetry Eight brand new files, including two from each of the reciprocating file systems were subjected for DSC testing. To be able to investigate differences in thermal behavior of the apical and coronal portions of the rotary files, the files were separated into two fragments using a diamond bur under copious water coolant. The apical fragments, consisting of D0-D4 of the rotary file, and the coronal segments, consisting of D7-D11 of the rotary file, were individually assessed.  The DSC2500 machine (TA Instruments, New Castle, USA) was used in this study (Figure 9 and Figure 10). Each fragment was weighed with an electronic balance and was then placed in a pre-weighed aluminum cell that consisted of a Tzero pan and a Tzero Hermetic Lid (TA Instruments, New Castle). The reference sample was an empty Tzero pan sealed with a Tzero Hermetic Lid. With the use of liquid nitrogen, the sample was initially cooled to -75oC. When the sample reached -75oC, it was then heated at 10oC/minute until it reached 100oC. After a brief pause, the sample was then re-cooled to -75oC at a rate of 10oC/min. The data was transferred to TRIOS software (TA Instruments, New Castle, USA), which was used to analyze the thermal behavior of each specimen. The phase transformation temperatures (Ms, Mf, As, Af and Rs) were determined on the thermograms by using the TRIOS software function “Onset Point”. This function uses a tangent line where the phase transformation curves begin to deviate from the adjacent baseline in order to place a point at the temperature that the phase transformation is most likely to occur (Bradley et al. 1996, Miyai et al. 2006).   22   Figure 9: Photograph of the DSC2500 machine.   Figure 10: Photograph of DSC2500 tray containing samples that are sealed in aluminum cells. 23  2.4 Statistical analysis Statistical analysis was performed with SPSS software version 3.1.1.0 (IBM, Armonk, New York, USA). The normality of the data was determined by using the Kolmogorov-Smirnov test. The equality of variance was assessed with Levene’s test. One-way analysis of variance (ANOVA) was used to compare the average NCF of the study groups. Post hoc multiple comparison with the Tukey test was used to identify and confirm statistically significant differences between groups. The Kruskal-Wallis test was used to compare the mean fragment lengths between groups. The Mann Whitney U test was used to compare the means of the phase transformation temperatures between the apical and coronal fragments for each file system. The statistical significance level, α, was set at 0.05. 24  Chapter 3: Results 3.1 Cyclic fatigue test The NCF for the reciprocating files are displayed in Table 1. Graphic comparisons of the NCF between the reciprocating file systems are presented in Figure 11 and Figure 12.  3.1.1 Differences based on file system As the CFR of a file is representative of the number of cycles the file experiences before fracture occurs, CFR will be used interchangeably with the NCF. The only significant difference in CFR between WOG and REB occurred when the canal’s DOC was 11 mm (p < 0.05). REB had significantly greater CFR than WO and RE in all groups (p < 0.05). WOG had significantly greater CFR than WO when used in a canal with a DOC of 5 mm, 8 mm, 10 mm, and 11 mm    (p < 0.05) while no differences existed between these files when the canal’s DOC was 6 mm     (p > 0.05). WOG had significantly greater CFR than RE in canals with a DOC of 5 mm, 8 mm, and 10 mm (p < 0.05) while there were no differences between these files when used in canals with a DOC of 6 mm or 11 mm (p > 0.05). Compared to WO, RE had superior CFR in the canal with a DOC of 11 mm (p < 0.05).  3.1.2 Differences based on canal curvature location All the reciprocating files showed significantly decreased CFR when they were used in canals with a DOC of 5 mm, 6 mm, and 8 mm when compared to canals with a DOC of 10 mm or 11 mm (p < 0.05). Additionally, WO, RE, and REB showed significantly increased CFR when used in a canal with a DOC of 11 mm when compared to a canal with a DOC of 10 mm (p < 0.05). 25  There were no significant differences in CFR when WOG was used in canals with a DOC of 10 mm or 11 mm (p > 0.05).    Mean NCF + SD Canal type WO WOG RE REB Group 1 75 + 22 1 a 239 + 91 2 a 84 + 31 1 a 158 + 60 2 a Group 2 98 + 24 1 a 193 + 61 1 2 a 110 + 33 1 a 220 + 126 2 a Group 3 89 + 28 1 a 293 + 101 2 a 124 + 24 1 a 318 + 103 2 a Group 4 327 + 78 1 b 799 + 136 2 b 289 + 64 1 b 718 + 144 2 b Group 5 473 + 173 1 c 902 + 136 2 b 677 + 138 2 c 1077 + 310 3 c Table 1: Number of cycles to failure (NCF). Statistically significant differences (p < 0.05) exist between groups in a row that have different superscript numbers, and when values in a column have different subscript letters. Canal groups: 1, 2, 3, 4, and 5 represent 5 mm, 6 mm, 8 mm, 10 mm, and 11 mm distance between the canal orifice and the curvature location (DOC), respectively.         26   Figure 11: Comparison of the mean NCF between different reciprocating NiTi rotary files when used in artificial canals with different curvature locations. Statistically significant differences (p < 0.05) exist between groups with different letters.   Figure 12: Effect of canal curvature location on the mean NCF of reciprocating NiTi rotary files. Statistically significant differences (p < 0.05) exist between groups with different letters. a a  b b a ab a b a b a b a  b a b a b b c   a b c a b b a b c  a b c a a a a  a a a a 27  3.1.3 Fractured fragment length The mean fractured fragment length values are displayed in Table 2 and are arranged as graphs for visual comparison in Figure 13 and Figure 14.  There were no significant differences in length of the fractured fragments between the reciprocating file systems (p > 0.05) except between WOG and RE in the canal with a DOC of 11 mm (p < 0.05). Based on the mean length of the file fragments, the file fractures occurred at or near the respective canal’s point of maximum curvature.   Mean fragment length + SD (mm) Canal type WO WOG RE REB Group 1: 5mm 9.5 + 0.2 a b c 9.7 + 0.3 b 9.3 + 0.3 c 9.5 + 0.5 a b c Group 2: 6mm 8.7 + 0.6 a 8.7 + 0.8 a 8.3 + 0.3 a 8.5 + 0.5 a Group 3: 8mm 6.2 + 0.5 a 6.4 + 0.4 a 6.1 + 0.3 a 6.5 + 0.3 a Group 4: 10mm 4.3 + 0.5 a 4.2 + 0.1 a 4.4 + 0.3 a 4.3 + 0.3 a Group 5: 11mm 3.8 + 0.6 a 3.8 + 0.3 a 3.4 + 0.2 a 3.5 + 0.2 a Table 2: The length of the fractured fragments in millimeters. Statistically significant differences (p < 0.05) exist between groups in the same row with different subscript letters.    28   Figure 13: Comparison of the mean fractured fragment length between different reciprocating NiTi file systems in artificial canals with different canal curvature locations. Statistically significant differences (p < 0.05) exist between groups with different letters.   Figure 14: Effect of the canal curvature location on the mean fractured fragment length of reciprocating NiTi reciprocating file systems a a a a abc b c abc a a a a  a a a a a a a a 29  3.1.4 Fractographic examination The surfaces of the fractured file fragments all showed evidence of fatigue failure, including signs of crack initiation, fatigue striation lines, and dimpling/microvoid formation (Pruett et al. 1997). The crack initiation points were seen at stress concentration areas. The high magnification images typically showed crack initiation at the file’s cutting edges with clusters of fatigue striations. SEM fractographs of the surface of the fractured file fragments can be seen in Figure 15Figure 22.    30   Figure 15: SEM fractograph (x150) of a WO file fragment. Crack initiation points are shown by white arrows.  Figure 16: Increased magnification (x600) of Figure 15. Crack initiation points are shown by white arrows and fatigue striations are shown by black arrows. 31   Figure 17: SEM fractograph (x250) of a WOG file fragment. Crack initiation points are shown by white arrows.  Figure 18: Increased magnification (x650) of Figure 17. Crack initiation points are shown by white arrows. 32   Figure 19: SEM fractograph (x130) of a RE file fragment. Crack initiation points are shown by white arrows.  Figure 20: Increased magnification (x550) of Figure 19. Crack initiation points are shown by white arrows and fatigue striations are shown by black arrows. 33   Figure 21: SEM fractograph (x130) of a REB file fragment. Crack initiation points shown by white arrows.  Figure 22: Increased magnification (x500) of Figure 21. Crack initiation points shown by white arrows and fatigue striations are shown by black arrows. 34  3.2 Differential scanning calorimetry The phase transformation temperatures acquired during the DSC cooling phase (Ms and Mf) of the file fragments are presented in Table 3. The phase transformation temperatures acquired during the DSC heating phase (Rs, As and Mf) of the file fragments are presented in Table 4. There were no significant differences in any of the phase transformation temperatures between the apical and coronal fragments of the same file system (p > 0.05). The coronal and apical fragments of all file systems exhibited a similar pattern of thermal behavior as noted by visual assessment of the DSC thermograms. An example of a DSC thermogram comparing the apical and coronal fragments is depicted in Figure 23. By combining the apical and coronal fragment groups, the mean phase transformation temperatures for each file system were determined (Table 5).  The DSC thermograms shown in this study include a cooling curve at the top of the figure and a heating curve at the bottom of the figure. The thermograms for WO, WOG, and RE exhibited single peaks in both the heating and cooling directions (Figure 24Figure 26). This suggests that WO, WOG, and RE undergo a one-step phase transformation from martensite to austenite during heating and a one-step phase transformation from austenite to martensite during cooling. The thermograms for REB exhibit a single exothermic peak in the cooling direction but double endothermic peaks in the heating direction (Figure 27). It appears that REB undergoes a one-step phase transformation from austenite to martensite when cooled while a two-step phase transformation from martensite to R-phase to austenite takes place when the file is heated. The phase transformation peaks associated with WO and RE were broad while sharp peaks were 35  noted with REB. Figure 28 depicts a DSC thermogram comparing the thermal behavior of the different reciprocating file systems.   Phase Transformation Temperatures During Cooling + SD System - Fragment Ms (oC) Mf  (oC) WO – Apical 46.26 + 0.42 21.86 + 1.11 WO - Coronal 43.94 + 0.36 23.22 + 0.01 WOG – Apical 44.14 + 0.01 26.92 + 0.60 WOG – Coronal 44.65 + 0.08 25.59 + 0.93 RE – Apical 44.46 + 0.62 24.48 + 3.25 RE – Coronal 45.32 + 0.32 22.96 + 0.52 REB – Apical 31.98 + 1.10 24.86 + 0.63 REB - Coronal 33.13 + 0.68 23.32 + 1.04 Table 3: Mean phase transformation temperatures + standard deviation acquired by cooling the apical and coronal fragments of the reciprocating file systems. Ms = martensite transformation start temperature. Mf = Martensite transformation finish temperature. There were no significant differences in Ms or Mf between the apical and coronal fragments of each file system (p > 0.05).       36  Phase Transformation Temperatures During Heating + SD System - Fragment Rs (oC) As (oC) Af (oC) WO – Apical N/A 24.74 + 1.02 49.49 + 2.01 WO – Coronal N/A 27.70 + 0.35 47.90 + 1.34 WOG – Apical N/A 35.98 + 0.57 46.78 + 0.06 WOG – Coronal N/A 34.73 + 0.08 47.74 + 1.48 RE – Apical N/A 27.60 + 1.29 50.86 + 2.47 RE – Coronal N/A 25.91 + 0.16 52.00 + 0.66 REB – Apical 13.28 + 0.87 30.32 + 0.32 36.60 + 0.39 REB - Coronal 12.14 + 0.57 30.16 + 0.12 37.56 + 0.13 Table 4: Mean phase transformation temperatures + standard deviation acquired by heating the apical and coronal fragments of the reciprocating file systems. Rs = R-phase transformation start temperature. As = Austenite transformation start temperature. Af = Austenite transformation finish temperature. There were no significant differences in Rs, As or Af between the apical and coronal fragments of each file system (p > 0.05).  Combined Mean Phase Transformation Temperatures + SD File system Ms (oC) Mf (oC) Rs (oC) As (oC) Af (oC) WO 45.10 + 1.38 22.54 + 1.01 N/A 26.22 + 1.82 48.70 + 1.67 WOG 44.40 + 0.30 26.26 + 1.00 N/A 35.36 + 0.80 47.26 + 1.02 RE 44.88 + 0.64 23.72 + 2.09 N/A 26.76 + 1.23 51.42 + 1.62 REB 32.55 + 1.00 24.09 + 1.23 12.71 + 0.90 30.24 + 0.22 37.08 + 0.60 Table 5: Mean phase transformation temperatures + standard deviation of the reciprocating file systems when the coronal and apical fragments were combined into one group. 37   Figure 23: DSC thermogram comparing the apical and coronal fragments of WO.   Figure 24: DSC thermogram of a WO coronal fragment. Comparison Between the Apical and Coronal Fragments of WaveOneExo UpWaveOne Coronal FragmentExo UpMf = 23.21 °CMs = 43.68 °CAf = 46.95 °CAs = 27.95 °C38   Figure 25: DSC thermogram of a WOG coronal fragment.   Figure 26: DSC thermogram of a RE coronal fragment. WaveOne Gold Coronal FragmentExo UpMf = 24.93 °CMs = 44.59 °CAs = 34.86 °C Af = 46.70 °CReciproc Coronal FragmentExo UpMf = 23.33 °C Ms = 45.09 °CAf = 52.47 °CAs = 24.95°C39   Figure 27: DSC thermogram of a REB coronal fragment.   Figure 28: DSC thermogram comparing the coronal fragments of WO, WOG, RE, and REB. Reciproc Blue Coronal FragmentExo UpRs = 11.73 °CAf = 37.46 °CAs = 30.08 °CMf = 22.59 °C Ms = 32.65 °Cwoc2Exo Up40  Chapter 4: Discussion As heat-treatment is known to increase the flexibility and CFR of NiTi files, it is anticipated that WOG and REB would take longer to fracture in the cyclic fatigue test compared to their predecessors, WO and RE, respectively (Kuhn & Jordan 2002, Yahata et al. 2009). While past studies have demonstrated improved CFR in WOG and REB compared to WO and RE, they tend to use canals that have only apical curvatures (Ozyurek 2016, Keskin et al. 2017, Al-Obaida et al. 2019, Keles et al. 2019, Scott et al. 2019). There are few studies in the literature that compare the CFR of files rotating in canals with differing curvature locations. Two studies compared the CFR of continuously rotating files in a canal with an apical curvature versus a canal with a mid-root curvature (Lopes et al. 2011, Lopes et al. 2013). These studies concluded that BioRace and Mtwo had significantly reduced CFR in canals with a mid-root curvature than canals with an apical curvature (Lopes et al. 2011, Lopes et al. 2013). A study by Arias et al. compared the CFR of WO and RE in canals with a curvature located at 5 mm or 13 mm from the tip of the instrument and concluded that the more coronally-located curvature (at 13 mm from the tip of the instrument) significantly decreased the CFR of these reciprocating files (Arias et al. 2012). Apart from the three studies mentioned, there appears to be an absence of literature regarding the effect of canal curvature location on the CFR of rotary files. Furthermore, there does not seem to be any research studying the effect that different canal curvature locations can have on the CFR of modern heat-treated rotary files. In this study, REB had superior CFR compared to RE in all of the artificial canals while WOG had superior CFR compared to WO in all canals except for the canal with a DOC of 6 mm. Differences in CFR between rotary files may be due to differences in diameter, taper, metallurgy, 41  and cross-section of the rotary file (Pruett et al. 1997, Haïkel et al. 1999, Yahata et al. 2009). Both RE-25 and REB-25 files have the same geometric designs, including an S-shape cross-section, a variable taper, a D0 tip diameter of 0.25 mm and apical taper (D0-D3) of 0.08 mm. As the only difference between RE and REB is their metallurgy, it should be safe to say that the heat-treatment of REB resulted in its comparatively superior CFR. Apart from the same D0 tip size of 0.25 mm and a variable taper along the file length, there are significant differences between the WO and WOG primary files. In order to improve dentin conservation, WOG was made with a 0.07 mm apical taper while WO was manufactured with a 0.08 mm apical taper. WO has two cross-sectional designs, a modified convex triangle from D0-D8 and a convex triangle from D9-D16 while WOG has a parallelogram cross-section. This study is unable to determine whether any or all the differences between WO and WOG primary files, including metallurgy, taper, or cross-section, are responsible for their differences in CFR. The reciprocating files in this study experienced significantly reduced CFR when operated in canals with a DOC of 5 mm, 6 mm, and 8 mm compared to those with a DOC of 10 mm and 11 mm. In the current study, DSC was used to compare the thermal behavior between the apical and coronal fragments of each file system. There were no significant differences in the phase transformation temperatures between the apical and coronal fragments of any of the tested file systems. Therefore, variances in crystal structure due to differences in thermal behavior along the length of the file does not seem to be a likely cause for the decreased CFR the file experiences when reciprocating in a coronally-located canal curvature.  In order to improve on this study, future studies may wish to compare differences in CFR in coronally-located 42  curvatures between continuous rotation and reciprocating files, and files with different tapers, diameters, cross-sections, and metallurgies. It is important for fatigue resistance studies to design an experimental apparatus that simulates root canal treatment so that the results of the study are clinically relevant and useful for clinicians to improve endodontic treatment. Irrigants are used in CFR studies not only to reduce heat generation caused by friction between the reciprocating file and the artificial canal, but also to control the environmental temperature, which can have a substantial effect on the crystal structure and CFR of the rotary file (Otsuka & Ren 2005, Shen et al. 2009, Alfawaz et al. 2018, Keles et al. 2019). An in vivo study by Hemptinne et al. used thermocouple microprobes during root canal treatment and found that intracanal sodium hypochlorite would rapidly equilibrate to 35 + 1oC (Hemptinne et al. 2015). The current study intended to replicate intracanal temperature by maintaining the deionized water that filled and surrounded the artificial canals at a constant temperature of 37 + 2oC. Sodium hypochlorite is the most popular irrigant that is used in endodontic treatment due to its lubricating, antimicrobial and tissue-dissolving qualities (Siqueira et al. 1997, Jeansonne & White 2006, Haapasalo & Shen 2010, Stojicic et al. 2010). As sodium hypochlorite is corrosive to metal, it has been speculated that sodium hypochlorite may reduce the CFR of NiTi instruments. With the use of SEM, Cheung et al. was able to identify corrosion pits on the periphery of NiTi file fragments that suffered fatigue failure in a sodium hypochlorite environment (Cheung et al. 2007). Alfawaz et al. showed that sodium hypochlorite, especially at full-strength concentrations, significantly decreased the CFR of NiTi rotary files compared to distilled water (Alfawaz et al. 2018). There are numerous studies regarding sodium hypochlorite’s influence on the CFR of rotary files, but the results have been controversial. 43  Studies by Keles et al. and Cheung et al. agreed with Alfawaz et al., while Elnaghy and Elsaka, Huang et al., and Pedulla et al. found that sodium hypochlorite had no significant effect on the CFR of rotary files (Cheung et al. 2007, Pedulla et al. 2013, Pedulla et al. 2014  ¸Elnaghy & Elsaka 2016  ¸Huang et al. 2017, Alfawaz et al. 2018, Keles et al. 2019). Deionized water was chosen as the lubricating medium in this study as the addition of another variable (e.g. sodium hypochlorite as a second lubricating medium) would have resulted in decreased power of the study due to the limited number of files available for use. It is possible that the results of this study may overvalue the CFR of these reciprocating rotary files when compared to their clinical use with sodium hypochlorite as an irrigant. The most important temperature-based characteristic of NiTi rotary files during clinical endodontic treatment is the potential phase transformation of martensite to austenite. To achieve maximum possible CFR, the NiTi file would need to have an As and Af above 35 + 1oC, which is the clinical intracanal temperature (Hemptinne et al. 2015). If the As and/or Af values of the NiTi are between room temperature (approximately 23oC) and the clinical intracanal temperature, the file will undergo phase transformation when used in the root canal. The clinician would most likely not notice the shift from martensite to austenite, which results in increased rigidity and decreased CFR of the file and can result in an increased risk of fatigue failure in canals with curvatures (Shen et al. 2013). The reciprocating files used in this study had mean As values that ranged from 26.22 to 35.36 oC and Af values that ranged from 37.08 to 51.42oC (Table 5). It would be reasonable to assume that the heat-treated files, WOG and REB, have high As and Af values as their superior flexibility and fatigue resistance over WO and RE is suggestive of martensitic alloy (Miyai et al. 2006, Hieawy et al. 2015). WOG fulfills this assumption as it 44  should be almost completely in the martensitic phase with As. and Af values of 35.36oC and 47.26oC, respectively, if intracanal temperatures of 35 + 1oC are to be assumed (Hemptinne et al. 2015). In a recent paper, Garcia et al. used DSC and X-ray diffraction to analyze WO and WOG files (Garcia et al. 2019). In addition to presenting similar phase transformation values for WO and WOG as the current study, they also found the presence of austenite and R-phase in WO and WOG at room temperature (Garcia et al. 2019). Unfortunately, due to peak superposition, Garcia et al. were not able to determine the amount of each phase present in these files at room temperature (Garcia et al. 2019). While it would be interesting to know how heat-treatment affects the phase composition of WOG, especially at intracanal temperature, it appears that this paper by Garcia et al. is currently the only available study in the literature regarding this topic (Garcia et al. 2019). While REB had a higher As (30.24oC) than WO and RE, it also had the lowest Af of all the tested files in this study with a value of 37.08oC. Based on these results, it would seem likely that REB would be mostly austenite during clinical root canal treatment, which is unexpected due to its superior CFR compared to WO and RE, which have higher Af values. Chi et al. used DSC and fatigue resistance testing on ProTaper Universal (PTU) F2 files that either had no heat-treatment (control), were heat-treated at 400oC, or were heat treated at 600oC in a nitrate bath (Chi et al. 2017). They found that PTU files heat-treated at 600oC had the best CFR followed by heat-treatment at 400oC and finally the control group (Chi et al. 2017). Interestingly, their DSC results showed that the 400oC heat-treatment group had elevated phase transformation temperatures compared to the control group while the 600oC heat-treatment group had reduced phase transformation temperatures compared to the control group (Chi et al. 2017). Chi et al.’s study 45  results are akin to the findings in the current study where REB, a file that differs only in heat-treatment, has superior CFR yet reduced Af values relative to RE. The current study’s thermogram for REB consists of sharp peaks, which are characteristic of NiTi with a high R-phase composition, while broad peaks, which are characteristic of a high-austenite composition, can be seen with thermograms regarding WO, WOG, and RE (Figure 28) (Garcia et al. 2019, Generali et al. 2019). This is consistent with the fact that REB has a two-stage phase transformation upon heating, which is indicative of the presence of R-phase (Figure 27). With the use of x-ray diffraction, Generali et al. found that the major phase composition of REB was R-phase and austenite while small amounts of martensite were present at room temperature (Generali et al. 2019). In the same study, they found that RE was primarily austenite while R-phase and martensite could also be detected in the samples (Generali et al. 2019). While elevations in phase transformation temperatures are an obvious benefit of heat-treatment of NiTi rotary files, these findings suggest that alteration of the phase composition of these files may be the most significant benefit of heat-treatment when considering CFR.   46  Chapter 5: Conclusion Reciprocating rotary files experience decreased CFR in canals with middle- or coronally-located curvatures when compared to canals with apically-located curvatures. WOG and REB have superior CFR when compared to WO and RE. When heated, WO, WOG, and RE undergo a one-stage phase transformation from martensite to austenite, while REB undergoes a two-stage phase transformation from martensite to R-phase to austenite.   47  Bibliography  Al-Obaida M, Merdad K, Alanazi M et al. (2019) Comparison of cyclic fatigue resistance of 5 heat-treated nickel-titanium reciprocating systems in canals with single and double curvatures. Journal of Endodontics 45, 1237–41. 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